This invention relates to a method of sizing liposomes, and more particularly to a sizing method which includes extruding liposomes through a branched-pore type aluminum oxide porous film.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic xe2x80x9ctailxe2x80x9d region and a hydrophilic xe2x80x9cheadxe2x80x9d region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) xe2x80x9ctailsxe2x80x9d of the lipid monolayers orient toward the center of the bilayer while the hydrophilic xe2x80x9cheadxe2x80x9d orient towards the aqueous phase.
The original liposome preparation of Bangham, et al. (J. Mol. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to xe2x80x9cswellxe2x80x9d, and the resulting liposomes, which consist of multilamellar vesicles (MLVs), are dispersed by mechanical means. This technique provided the basis for the development of the small sonicated unilamellar vesicles (SUVS) described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), as well as large unilamellar vesicles (LUVs). In addition, U.S. Pat. No. 4,235,871, issued Nov. 25, 1980 to Papahadjopoulos et al., describes a xe2x80x9creverse-phase evaporation processxe2x80x9d for making oligolamellar lipid vesicles, also known as reverse-phase evaporation vesicles (REVs).
Alternative methods have been developed for forming improved classes of multilamellar vesicles which have been shown to have particularly improved properties such as, for example, higher active ingredient trapping efficiencies and loadability, better stability, less leakage, and greater ease of production. One such improved class of liposomes, denominated as stable plurilamellar vesicles (SPLVs), is described in U.S. Pat. No. 4,522,803, issued Jun. 11, 1985 to Lenk et al. Another such improved class, defined as monophasic vesicles (MPVs), is described in U.S. Pat. No. 4,558,578, issued May 13, 1986 to Fountain et al. Both of these classes of liposomes have also been characterized as having substantially equal interlamellar solute distributions. A general review of various methods for producing liposomes, including an extensive bibliography, is set forth in Deamer and Uster, xe2x80x9cLiposome Preparation: Methods and Mechanismsxe2x80x9d, in the Liposomes, edited by M. Ostro, pp. 27-51 (1983), incorporated herein by reference.
The administration of drugs encapsulated in or otherwise associated with liposomes has been proposed for use in a variety of drug delivery regimens in combination with or as an alternative to the administration of free drugs. In some applications, liposomes have been found to provide sustained release of drugs for extended periods, which can be of particular importance in the lengthy chemotherapy regimens often required for the treatment of various forms of cancer or AIDS-related illnesses. Another property of liposomes is their ability to be taken up by certain cells, such as phagocytes, such that they can deliver their active ingredient to the interior of the cells. This makes such liposome treatment particularly useful in treating intracellular infections, such as those associated with species of Mycobacteria, Brucella, Listeria, and Salmonella. Thus, drugs encapsulated in liposomes can be delivered for the treatment of such intracellular diseases without administering large amounts of free unencapsulated drug into the bloodstream. In addition, the mere association of certain drugs or other bio-active agents with liposomes has been found to potentiate or improve the activity of such drugs or bio-active agents, or to reduce their toxicity.
Liposomes behave like particles, and are commonly described in terms of average particle size and particle-size distributions. For certain uses of liposomes, particularly in the parenteral administration of drugs, it is important to size the liposomes to a desired average particle size, and to maintain a controlled particle-size distribution, particularly by sizing the liposomes so that substantially all of the liposomes are of a size below a predetermined maximum diameter. For liposomes intended for parenteral administration, one desirable size range is between about 100 and 1000 nm, preferably between about 100 and 500 nm. (As used herein, nm represents nanometer (10xe2x88x929 m) and um represents micrometer or micron (10xe2x88x926 m).) The maximum desired size range is often limited by the desire to sterilize the liposomes by filtering through conventional sterilization filters, which commonly have a particle-size discrimination of about 200 nm. However, overriding biological efficacy and/or safety factors may dictate the need for a particular particle size, either larger or smaller. Control of the size range of the liposomes may also improve the effectiveness of the liposomes in vivo, as well as the stability and leakage resistance of the liposomes.
The various methods for producing liposomes generally produce a suspension of liposomes of widely varying sizes, many of which exceed 1000 nm in average particle size. A number of methods have been proposed to reduce the size and size distribution of liposomes in such suspensions. In a simple homogenization method, a suspension of liposomes is repeatedly pumped under high pressure through a small orifice or reaction chamber until a desired average size of liposome particles is achieved. A limitation of this method is that the liposome size distribution is typically quite broad and variable, depending on the number of homogenization cycles, pressures, and internal temperature.
Small unilamellar vesicles (SUVs), generally characterized as having diameters below 100 nm, are composed of highly strained, curved bilayers. The SUVs are typically produced by disrupting larger liposomes via ultrasonication. It has been found that a narrow size distribution of such liposomes can only be achieved when the liposomes have been reduced to their smallest sizes, less than about 50 nm. Furthermore, this process may not be amenable to large-scale production, because it is generally conducted as a batch process with long-term sonication of relatively small volumes. In addition, heat build-up during sonication can lead to peroxidative damage to lipids, and sonication probes may shed titanium particles which are potentially quite toxic in vivo.
A method of sizing liposomes by filtration through a 200-nm Unipore(trademark) polycarbonate filter is discussed in Szoka, Proc. Natl. Acad. Sci. U.S.A., 75:4194-8 (1978). A size-processing method based on liposome extrusion through a series of uniform straight-pore type polycarbonate membranes from about 1000 nm down to about 100 nm is described in Hunt et al., U.S. Pat. No. 4,529,561, issued Jul. 16, 1985. However, this method can be relatively slow, often requiring many passes through various size filters to obtain the desired particle-size distribution.
Vesicles may also be size-reduced using an extrusion process described in Cullis et al., U.S. Pat. No. 5,008,050, issued Apr. 16, 1991, incorporated herein by reference. Vesicles made by this technique are extruded under pressure through a filter with a pore size of 100 nm or less. This procedure avoids the problems of the above homogenization and sonication methods, and does not require multiple passes through decreasing size filters, as described in the above-cited U.S. Pat. No. 4,529,561. Such a process can provide size distributions of liposomes that are quite narrow, particularly by cycling the material through the selected size filter several times. In addition, it is believed that such extrusions may convert multilamellar vesicles into oligolamellar or even unilamellar form, which may be desired for certain applications. However, as demonstrated by the Examples set forth below in the present specification, when such extrusions are made through 100-nm polycarbonate filters, such as the Nuclepore(trademark) filters used in the examples of this reference, even at relatively high pressures flow rates may be relatively low.
U.S. Pat. No. 4,737,323, issued Apr. 12, 1988, describes a method for sizing liposomes by extrusion through an asymmetric ceramic filter. Such filters are designed for operation at relatively high pressure, and can be backflushed to prevent clogging. U.S. Pat. No. 4,927,637, issued May 23, 1990 describes a method of sizing liposomes by passing them through a polymer filter having a web-like xe2x80x9ctortuous-pathxe2x80x9d construction.
An alternative type of filter medium is described in Furneaux et al., U.S. Pat. No. 4,687,551, issued Aug. 18, 1987. This patent discloses a new type of filter sheet comprising an anodic aluminum oxide film having branched pores extending from one surface of the film to the other. The film is unique in that it includes a system of larger pores extending in from one face and a system of smaller pores extending in from the other face. The system of larger pores interconnects with the system of smaller pores such that the inner ends of one or more smaller pores are joined to the inner end of a larger pore and there are substantially no blind larger pores. This patent is incorporated by reference into the present specification for the purpose of disclosing such branched-pore type aluminum oxide porous films and the method for forming them.
In a particular embodiment, the branched-pore anodic aluminum oxide film of the Furneaux et al. patent is described as:
An anodic aluminum oxide film having pores extending from one face of the film to the other,
including a system of larger pores extending in from one face a distance into the film, the larger pores having a diameter d near their inner ends, and a system of smaller pores extending in from the other face a distance s into the film, the smaller pores having a substantially uniform minimum diameter p,
the system of larger pores interconnecting with the system of smaller pores, such that the inner ends of one or more smaller pores are joined to the inner end of a larger pore and there are substantially no blind larger pores, wherein
d is 10 nm to 2 um
p is at least 2 nm but less than 0.5 d, and
s is 10 nm to 1 um.
The size rating of such branched-pore type films is equal to p, the substantially uniform minimum diameter of the smaller pores.
Filtration membranes made in accordance with the disclosure of the Furneaux et al. patent are commercially available and sold by the Anotec Separations, New York, N.Y., under the name Anopore(trademark). Additional information regarding such branched-pore type membranes is provided in Furneaux et al., xe2x80x9cThe Formation of Controlled-Porosity Membranes from Anodicaily Oxidized Aluminumxe2x80x9d, Nature 337:147-9 (1989).
One use of such branched-pore Anopore(trademark) filters is described in Jones et al., xe2x80x9cComparison of a New Inorganic Membrane Filter (Anopore) with a Track-Etched Polycarbonate Membrane Filter (Nuclepore) for Direct Counting of Bacteriaxe2x80x9d, Applied and Environmental Microbiology 55(2):529-30 (1989). This article compares the bacteria filtering ability of a 200-nm-pore-size Anopore(trademark) filter against a 200-nm-pore-size Nuclepore(trademark) filter.
In accordance with the method of the present invention, a population of liposomes substantially free of liposomes above a predetermined maximum size is produced by (1) providing a suspension of liposomes, a portion of which are of sizes larger than the predetermined maximum size; and (2) passing the suspension under pressure one or more times through an aluminum oxide porous film.
Films with a pore size of 1000 nm or less may be used to obtain liposomes with an average particle size of in the range of about 100 to 1000 nm. In a particular embodiment of the present invention, a film with a pore-size rating of 200 nm or less is used to obtain a population of liposomes with a predetermined maximum diameter of less than about 500 nm. In another embodiment, a film with a Pore size of about 100 nm or less is used, and the suspension of liposomes is passed through the filter one or more times until the average liposome particle size is about 100 to 200 nm.
In a further embodiment of the present invention, the suspension of liposomes is passed repeatedly through the porous film until a desired particle size distribution is obtained. In a particular embodiment, the liposomes are passed through the porous film two to ten times. In an additional embodiment, the liposomes are presized by being passed one or more times through a 2-10 micrometer filter.
A preferred film for use in the present invention is a branched-pore type anodic aluminum oxide porous film. As discussed above, such a branched-pore anodic aluminum oxide porous film is an aluminum oxide sheet having two substantially parallel major faces with pores extending from one face of the sheet to the other, including a system of larger pores extending from one face into the sheet and a system of smaller pores extending in from the other face, the system of larger pores interconnecting with the system of smaller pores such that the inner ends of one or more smaller pores are joined to the inner end of a larger pore and there are substantially no blind larger pores. The size rating of such branched-pore type films is equal to the minimum diameter of the smaller pores, which are preferably substantially uniform.
In a further embodiment of the present invention, apparatus is provided for carrying out the filtration method. The apparatus comprises one or more filter assemblies for holding the aluminum oxide porous films in operational configuration, means for supplying the suspension of liposomes to the filter assemblies, and means to receive the filtrate from the assemblies. In a particular embodiment, two or more assemblies are used in parallel configuration to filter the liposome suspension passing from the supply means to the receiving means. Optionally, means can also be provided for recirculating at least a portion of the filtrate from the receiving means back to the supply means, thus providing for multiple passes of the liposomes through the filters. In addition, a sterilization filter can be provided downstream of the receiving means, as may be appropriate to prepare the filtrate for pharmaceutical use.
The extrusion is rapid and inexpensive, and does not require the use of solvents or other chemicals that must be removed.